μNeurocircuitry: Establishing models of neurocircuits with human neurons.

Diagram of μNeurocircuitry device designed for interconnecting three different subtype neurons(a) Diagram of a brain nucleus receiving two inputs. (b) Device schematic. Four outer chambers surround one large central chamber. Axons project through 10 μm wide microchannels to communicate from the outer to central chamber. (c) Completed device bonded to glass cover slip. Colored dye indicates different chambers. (d) Fabrication protocol. Soft lithography was used to create a 3 μm layer followed by a 320 μm layer. PDMS is poured onto the master and cured. Holes are punched in the PDMS and the device is bonded to glass to yield the final device. (e) 3D representation of device with insets showing 3 μm channel height. (f) Bright field image of microchannels and corresponding dimensions.

Human neuronal culture within the microdevice(a) Induced neurons (iNs) cultured within a 200 μm walled device. (b) GFP-positive neurons cultured in a 500 μm walled device across from non-fluorescent iNs in the central chamber. (c–e) GFP-positive excitatory neurons exhibit extensive axonal projections to the central chamber and interact with TH-positive (red) DA-ergic neurons. Dashed area in (c) is showing in (d). Arrowhead showing a possible synaptic contact.

Morphological analysis(a) Excitatory neurons express pan-neuronal marker MAP2 and vGlut2, an excitatory marker. (b) Dopaminergic (DA) neurons express MAP2 and synapsin, and tyrosine hydroxylase (TH), indicative of a DA phenotype. (c) GABAergic neurons express GAD6, indicative of an inhibitory phenotype. (d) Excitatory neurons infected with GFP express pan-neuronal marker β-III-tubulin (Tuj1). (e) DAergic neurons co-localizing with synapsin-positive boutons. (f) Interaction of inhibitory (green) and excitatory (red) neurons with synapsin-positive boutons.

Three-way circuit connectivity among neurons cultured in different compartments(a) Two side chambers project axons into the central chamber. GFP-labeled induced neurons (iNs) were seeded on the right side and tdTomato-labeled iNs were seeded on the left. (b–c) Central chamber neurons interact with processes from both GFP and tdTomato-labeled side chamber iNs. These iNs stain positive for synapsin (blue) and MAP2 (white). (d) A MAP2-positive neuron receiving projections from both tdTomato-positive and GFP-positive axons. White arrowheads indicate synapsin-positive boutons.

Basal functional characterization of human neurons in the microdevice(a) Microdevice under electrophysiology patch clamp recording setup. (b) Spontaneous action potentials were recorded showing functional maturation of iNs. (c) Step current experiment performed on GABAergic neurons. They fire repetitive action potentials upon depolarization. Step size is 5 pA. (d) Whole-cell currents were recorded in these cells showing a fast activation and inactivation sodium currents and outward potassium currents. Step size is 10 mV. (e) Spontaneous post-synaptic currents recorded from central chamber (GABAergic neurons). (f) Spontaneous post-synaptic currents (PSCs) recorded from central chamber could be blocked by 20 μM CNQX indicating glutamatergic transmission.

Optogenetics-aided synaptic functional analysis for outer-to-center neurocircuitry(a) Device wall with microchannels. ChR2-tdTomato (red)-infected excitatory neurons project to β-III-tubulin-labeled inhibitory neurons (green). (b) Extensive excitatory projections inside the central chamber co-localize with MAP2-labeled neurons (green). Nuclei labeled with Hoechst (blue). Scale bar 50 μm. (c) Response to single pulse of blue light (470 nm). (d) Response to pulse train stimulation. (e) Post-synaptic currents (PSCs) recorded from central chamber neurons after excitation of terminals from projecting side chamber excitatory neurons with blue light. Arrow indicates asynchronous synaptic activity. (f) Addition of 20 μM CNQX eliminates AMPAR-mediated response to light pulse. Red traces show a single trace for visualization.